The long range goals of this research are to determine how the transmembrane domain sequences of "BH3-only"apoptotic regulatory proteins result in structural and functional interactions that give rise to programmed cell death. More than a dozen members of the 'BH3-only' subclass of the Bcl-2 superfamily provide cell- and signal-specific inputs to mitochondria-mediated apoptosis in mammals, but the biochemical and structural basis for this control is poorly understood. This project focuses on BNIP3 (Bcl-2/adenovirus E1B 19-kDa protein-interacting protein 3), a "BH3-only" protein that is functionally conserved from C. elegans to man. The transmembrane (TM) domain of BNIP3 is implicated in the pro-apoptotic function of the protein, in homodimerization, and in interactions with the anti-apoptotic protein Bcl-2. This research will determine the sequence specificity and structural basis for these TM domain interactions and will determine how these lateral associations within membranes contribute to the regulation of mitochondria-mediated apoptosis. Elucidating the mechanism of action of these proteins will have implications for our understanding of development, cancer, and neurodegenerative diseases. In the next five years, this proposed research will determine the structural basis for homo- and hetero-oligomerization of the hydrophobic C-terminal transmembrane (TM) domain of the mammalian "BH3-only" protein BNIP3 and its orthologs in C. elegans and D. melanogaster. Saturation mutagenesis experiments will define the sequence requirements for these associations in biological membranes and in detergents, and the structures of BNIP3 homodimers and heteromeric complexes will be determined using solution NMR spectroscopy. Interpretation of the mutagenesis data in the context of structures will elucidate the physical basis for the stability and specificity of protein-protein interactions inside membranes. Measuring the apoptogenic effects of wild type and mutant full-length BNIP3 constructs in vivo will determine the functional role(s) played by TM-TM interactions. The results will reveal fundamental rules of membrane protein folding, stimulate new types of experiments by cell biology researchers, and may identify novel drug targets for prevention of apoptosis-related cell death and organ damage such as hypoxia-induced apoptosis following ischemia.